The present invention, based on the discovery of a new biological phenomena, provides methods and compositions for use in identifying agents which modulate the interaction between Survivin and polymerized tubulin or the mitotic spindles. Related methods and compositions can be used to modulate the interactions between Survivin and polymerized tubulin or the mitotic spindles, thereby modulating Survivin regulated apoptosis.
Programmed cell death (sometimes referred to as apoptosis) is distinguishable, both morphologically and functionally, from necrosis. Programmed cell death is a natural form of death that organisms use to dispose of cells. Cells dying by programmed cell death usually shrink, rarely lyse, and are efficiently removed from the organism (rapidly recognized and engulfed by macrophages) without the appearance of inflammation (Michael Hengartner, xe2x80x9cCell Death and Aging, Molecular Mechanisms of,xe2x80x9d IN MOLECULAR BIOLOGY AND BIOTECHNOLOGY 158-62 (ed. R. A. Meyers, 1995)).
Apoptosis was initially used to describe a subset of programmed cell deaths sharing a particular set of morphological features which include membrane blebbing, shrinkage of cytoplasm, chromatic condensation and formation of a xe2x80x9cDNA ladder.xe2x80x9d During apoptosis, cells lose their cell junctions and microvilli, the cytoplasm condenses and nuclear chromatin marginates into a number of discrete masses. While the nucleus fragments, the cytoplasm contracts and mitochondria and ribosomes become densely compacted. After dilation of the endoplasmic reticulum and its fusion with the plasma membrane, the cell breaks up into several membrane bound vesicles, referred to as apoptotic bodies, which are usually phagocytosed by adjacent cells. Activation of particular genes such as tumor suppressor genes in vertebrates is thought to be necessary for apoptosis to occur. Apoptosis induced by numerous cytotoxic agents can be suppressed by expression of the gene bcl-2, which produces a cytoplasmic protein Bcl-2 (THE ENCYCLOPEDIA OF MOLECULAR BIOLOGY 67 (ed. John Kendrew et al., Blackwell Science; Oxford, England, 1994).
Survivin is a 142 amino acid protein (xcx9c16.5 kDa) that is expressed in tumor cells and embryonic tissues (C. Adida et al. (1998) Am. J. Pathol. 152(1): 43-9; and (1997) Gastroenterology 113(4): 1060). The gene is located on chromosome 17q25 and is a novel member of the IAP family of apoptosis inhibitors. The nucleic acid sequence which encodes Survivin is related to that of Effector Cell Protease Receptor-1 (EPR-1) but its orientation is assigned to the antisense EPR-1 strand.
Expression of Survivin in embryonic and fetal development may contribute to tissue homeostasis and differentiation that is independent of bcl-2 (Adida et al., 1998). Aberrations of this Survivin-associated developmental pathway may result in prominent re-expression of Survivin in neoplasia and abnormally prolonged cell viability (Adida et al., 1998).
Deregulation of programmed cell death has emerged as a primary mechanism contributing to the pathogenesis of various human diseases including cancer. While the impact of anti-apoptosis genes in neoplasia has focused, for example, on the role of bcl-2 in follicular lymphoma, a potential distribution of IAP proteins, such as Survivin, has only begun to been investigated. Survivin is rarely present in adult tissues but has been detected in adenocarcinoma of the pancreas, breast adenocarcinoma, colon cancer, head and neck squamous cell carcinoma, neuroblastoma, malignant thymoma, prostate cancer, and benign prostate hyperplasia (see U.S. Ser. No. 08/975,080). This expression pattern may suggest that overexpression of survivin or alterations in survivin gene regulation may commonly occur during tumorigenisis.
Living organisms are composed of cells, whose growth and division require a regular sequence of events and processes that comprise the cell cycle. Some cell cycle events are continuous (e.g., synthesis of proteins and lipids), whereas others are discontinuous (e.g., DNA synthesis). Two discontinuous processes for cell survival are the replication of DNA and the segregation of chromosomes to the daughters of cell division during mitosis. If either of these steps are performed inaccurately, the daughter cells will be different from each other and will almost certainly be flawed. Segregation of chromosomes occurs during mitosis, normally a relatively brief period in the cell cycle, which culminates in the highly visible act of cell division (e.g., cytokinesis). The remainder of cell cycle comprises interphase, during which growth occurs. Chromosome replication occurs in eukaryotic cells only during interphase; and replication and segregation are mutually exclusive processes.
Interphase is subdivided into the S phase when DNA synthesis occurs and the gaps separating S phase from mitosis. G1 is the gap after mitosis, before DNA synthesis starts; G2 is the gap after DNA synthesis is complete, before mitosis and cell division. Cellular constituents direct the cell cycle by acting as regulatory elements. In some cases, cellular proteins such as p53 act as xe2x80x9ccheck pointsxe2x80x9d or xe2x80x9cgate-keepersxe2x80x9d that promote or inhibit specific steps of the cell cycle.
The Mitotic Spindle and Tubulin
The mitotic spindle is a self-organizing structure that is constructed primarily from microtubules. Among the most important spindle microtubules are those that bind to kinetochores and form the fibers along which chromosomes move. These microtubules are comprised of xcex1-xcex2 tubulin dimers. xcex3-tubulin is a phylogenetically conserved component of microtubule-organizing centers that is essential for viability and microtubule function (T. Horio et al. (1994) J. Cell Biol. 126(6): 1465-73). It is exclusively localized at the spindle poles (also known as spindle pole bodies, SPB) in mitotic animal cells, where it is required for microtubule nucleation (M. A. Martin et al. (1997) J. Cell Sci. 110(5): 623-33; I. Lajoie-Mazenc et al. (1994) J. Cell Sci. 107(10): 2825-37). xcex3-tubulin is also found on osmiophilic material that lies near the inner surface of the nuclear envelope, immediately adjacent to the SPB (R. Ding et al. (1997) Mol. Biol. Cell 8(8): 1461-79).
One protein linked with the mitotic spindle is p53, which is a critical participant in a signal transduction pathway that mediates either a G1 arrest or apoptosis in response to DNA damage (S. E. Morgan et al. (1997) Adv. Cancer Res. 71:1-25). Loss of p53, in addition to suppression of apoptosis by bcl-2-related genes, may act cooperatively to contribute to genetic instability (A. J. Minn et al. (1996) Genes Dev. 10(2): 2621-31). The oncoprotein, Bcl-2, also has been demonstrated to be cell cycle specific, appearing in early prophase or late G2 and persisting throughout mitosis. The pattern of Bcl-2 localization shows a diffuse nuclear distribution before chromosome condensation, followed by a specific concentration of bcl-2 at the margins of condensed chromosomes in prophase, metaphase and anaphase (M. C. Willingham et al. (1994) J. Histochem. Cytochem. 42(4): 441-50).
Chemotherapeutics, such as taxol and the vinca alkaloids, perturb kinetochore-microtubule attachment and disrupt chromosome segregation. This activates a check point pathway that delays cell cycle progression and induces programmed cell death (P. K. Sorger et al. (1997) Curr. Opin. Cell. Biol. 9(6): 807-14; C. M. Ireland et al. (1995) Biochem. Pharmacol. 49(10): 1491-99). Taxol has been demonstrated to induce tubulin polymerization and mitotic arrest which is followed by apoptosis. Over expression of Bcl-x(L) in taxol induced cells has been demonstrated to interfere with the activation of a key protease involved in apoptosis (A. M. Ibrado et al. (1996) Cell Growth Differ. 7(8): 1087-94).
Although proteins that regulate apoptosis have been implicated in restraining cell cycle and controlling ploidy (chromosomal number), the effector molecules at the interface between cell proliferation and cell survival have remained elusive.
The present inventor has discovered that transcription of the survivin gene is cell cycle regulated and occurs exclusively in G2/M phase. This results in survivin re-distribution to the mitotic spindle where it mediates a default pathway of apoptosis inhibition in metaphase cells.
The present invention provides a method of identifying an agent which modulates one or more interactions between Survivin and tubulin comprising the steps of contacting Survivin and tubulin in the presence of the agent and determining whether the agent modulates one or more interactions between Survivin and tubulin, thereby identifying an agent which modulates one or more interactions between Survivin and tubulin.
The present invention further provides methods of identifying an agent which modulates one or more interactions of Survivin with the mitotic spindles of a cell, comprising the steps of contacting a mitotically active cell with the agent and determining whether the agent modulates one or more interactions of Survivin with the mitotic spindles of the cell, thereby identifying an agent which modulates one or more interactions of Survivin with the mitotic spindles.
The invention also provides methods of modulating the interaction between Survivin and tubulin comprising the step of administering an effective amount of an agent which modulates at least one interaction between Survivin and tubulin.
The invention further includes methods of modulating the interaction between Survivin and the mitotic spindles comprising administering an effective amount of an agent which modulates at least one interaction between Survivin and the mitotic spindles. The invention also provides methods of modulating apoptosis in a cell, comprising administering to the cell an effective amount of an agent which modulates the interaction between Survivin and tubulin. The invention further provides agents, compositions and peptides which modulate the interactions between Survivin and tubulin and/or the mitotic spindles.